[ Not Applicable ]
This invention relates to the field of chemically modified mutant enzymes. In particular this invention pertains to chemically modified mutant enzymes in which multiply charged substituents are introduced to enhance interaction of the enzyme with a charged substrate.
For both protein chemistry (Nilsson et al. (1992) Curr. Opin. Struct. Biol 2: 569-575; LaVallie and McCoy (1995) Curr. Opin. Biotechnol. 6: 501-506; Uhlen and Moks (1990) Methods Enzymol. 185: 129-143) and organic synthesis applications (Sears and Wong (1996) Biotechnol. Prog. 12: 423-433; Faber (1997) Biotransformations in Organic Synthesis: 3rd ed. Springer-Verlag: Heidelberg; Roberts (1993) Preparative Biotransformations; Wiley: New York: 1993) it is desirable to have available a diverse toolbox of inexpensive proteases with high selectivity and diverse substrate preferences. To date the most extensively exploited class of enzymes in organic synthesis applications have been the hydrolases. Among these, the serine proteases have received considerable attention due, in part, to their often exquisite stereo-, regio-, and chemo-selectivities (Sears and Wong (1996) Biotechnol. Prog. 12: 423-433; Faber (1997) Biotransformations in Organic Synthesis: 3rd ed. Springer-Verlag: Heidelberg; Roberts (1993) Preparative Biotransformations; Wiley: New York: 1993; Moree et al. (1997) J. Am. Chem. Soc. 119: 3942-3947).
While over 3000 enzymes have now been reported, of which many are proteases, significantly fewer of the latter are available inexpensively from commercial sources (Faber (1997) Biotransformations in Organic Synthesis: 3rd ed. Springer-Verlag: Heidelberg; Roberts (1993) Preparative Biotransformations; Wiley: New York: 1993; Moree et al. (1997) J. Am. Chem. Soc. 119: 3942-3947; Jones (1986) Tetrahedron 42: 3351-3403). Furthermore, since wild type enzymes do not accept all substrate structures of synthetic interest, it is attractive to contemplate the tailoring of a readily available protease in order to expand their substrate specificities in a controlled manner with the ultimate goal of creating any desired specificity at will.
In this regard, the goal of specificity alteration of enzymes has already been targeted by several different approaches. For example, site-directed mutagenesis (Perona and Craik (1995) Protein Sci. 4: 337-360) and random mutagenesis (Arnold (1998) Acc. Chem. Res. 31(3): 125-131) have been employed to tailor enzyme specificity and have permitted some insights into the electrostatic (Wells et al. (1987) Proc. Natl. Acad. Sci. USA, 84: 5167-5171; Wells et al. (1987) Proc. Nat. Acad. Sci. USA, 84: 1219-1223; Wells and Estell (1988) TIBS 13: 291-297; Bott et al. (1987) Pages 139-147 In: Biotech. Agric.Chem.; Lebanon, Mumma, Honeycutt, Duesing, eds.; Vol. ACS Symp. Ser. 334; Russell et al. (1987) J. Mol. Biol. 193: 803-813; Ballinger et al. (196) Biochemistry 33: 13579-13585), steric (Rheinnecker et al. (1994) Biochemistry 33: 221-225; Rheinnecker et al. (1993) Biochemistry 32(5): 1199-1203; Sxc3x8rensen et al. (1993) Biochemistry 32: 8994-8999; Estell et al. (1986) Science 233: 659-663; Takagi et al. (1996) FEBS Lett. 395: 127-132; Takagi et al. (1997) Protein Eng. 10(9): 985-989), and hydrophobic (Estell et al. (1986) Science 233: 659-663; Wangikar et al. (1995) Biochemistry 34(38): 12302-12310; Bech et al. (1993) Biochemistry 32: 2845-2852) factors which govern enzyme-substrate interactions. However, the structural variations within these approaches are limited to the 20-natural amino acids. Consequently, biosynthetic methods have recently been developed to introduce unnatural amino acids into proteins (25. Cornish et al. (1995) Angew. Chem. Int. Ed.Eng. 34: 621-633; Parsons et al. (1998) Biochemistry 37: 6286-6294; Hohsaka et al. (1996) J. Am. Chem. Soc. 118(40): 9778-9779). Unnatural functionalities have also been incorporated by chemical modification techniques (Kuang et al. (1996) J. Am. Chem. Soc. 118: 10702-10706; Ory et al. (1998) Protein. Eng. 11(4): 253-261; Peterson: E. B.; Hilvert: D. Biochemistry 34: 6616-6620; Suckling: C. J.; Zhu: L.-M. Bioorg. Med. Chem. Lett. 3: 531-534; Rokita and Kaiser (1986) J. Am. Chem. Soc. 108: 4984-4987; Kokubo et al. (1987) J. Am. Chem. Soc. 109: 606-607; Radziejewski et al. (1985) J. Am. Chem. Soc. 107: 3352-3354). Generally, however, unnatural amino acid mutagenesis approach is not yet amenable to large scale preparations, and chemical modification alone is insufficiently specific.
This invention provides novel multiply-charged chemically modified mutant enzymes. In a particularly preferred embodiment this invention provides a modified enzyme where one or more amino acid residues in the enzyme are replaced by cysteine residues. The cysteine residues are modified by replacing the thiol hydrogen in the residue with a substituent group providing a thiol side chain comprising a multiply charged moiety. Preferred enzymes include serine hydrolases, more preferably proteases (e.g. subtilisins). One particularly preferred enzyme is a Bacillus lentus subtilisin.
The amino acid replaced with a cysteine may include an amino acid selected from the group consisting of asparagine, leucine, methionine, and serine. Preferred replaced amino acids are in a binding site (e.g., a subsite such as S1, S1xe2x80x2, and S2). Where the enzyme is a subtilisin-type serine hydrolase the cysteine(s) is substituted amino acid(s) corresponding to a Bacillus lentus subtilisin residue selected from the group consisting of residue 156, reside 166, residue 217, residue 222, residue 62, residue 96, residue 104, residue 107, reside 189, and residue 209. Where the enzyme is a trypsin-chymotrypsin-type serine protease the cysteine(s) are substituted for and amino acid corresponding to a trypsin residue selected from the group consisting of Tyr94, Leu99, Gln175, Asp189, Ser190, and Gln192. Where the enzyme is an alpha/beta serine hydrolase the cysteine(s) are substituted for and amino acid corresponding to a Candida antartica lipase (protein Data Bank entry 1tca) residue selected from the group consisting of Trp104, Thr138, Leu144, Val154, Ile189, Ala225, Leu278 and Ile185.
The multiply charged moiety can be negatively or positive charged and in certain embodiments, the enzyme can contain both positively and negatively multiply charged moieties. Particularly preferred negatively charged moieties include, but are not limited to, sulfonatoethyl thiol, 4-carboxybutyl thiol, 3,5-dicarboxybenzyl thiol, 3,3-dicarboxybutyl thiol, and 3,3,4-tricarboxybutyl thiol, while particularly preferred positively charged moieties include, but are not limited to, amingethyl thiol, 2-(trimethylammonium)ethyl thiol, 4,4-bis(aminomethyl)-3-oxo-hexyl thiol, and 2,2-bis(aminomethyl)-3-aminopropyl thiol. The multiply charged moiety can also be a dendrimer or a polymer.
In another embodiment, this invention provides methods of making novel multiply-charged chemically modified mutant enzymes. The methods involve providing an enzyme having one or more amino acids have been replaced with cysteine residues; and replacing the thiol hydrogen, in one or more cysteine residues, with a substituent group providing a thiol side chain comprising a multiply charged moiety. In certain embodiments, a native cysteine can be chemically modified and there is no need to introduce a cysteine. Preferred enzymes include serine hydrolases as identified herein. Preferred residues for replacement with a cysteine and preferred multiply-charged moieties are identified herein.
In another embodiment, this invention includes a composition comprising any one of the multiply charged chemically modified mutant enzymes as described herein and a detergent or other cleaning agent.
In still another embodiment, this invention provides methods of assaying for a preferred enzyme. The methods involve providing a swatch of material comprising a piece of material and a stain; fixing the stain to the material; applying an enzyme to the swatch; and incubating the watch and the enzyme. The method can further involve determining the degree of removal of the stain from the material. Preferred enzymes for use in this method include, but are not limited to proteases, a cellulases, amylases, laccases, and lipases. In particularly preferred embodiments, the enzymes are modified serine hydrolases as described herein. Preferred materials include, but are not limited to fabrics, plastics, or ceramics. Preferred stains include, but are not limited to blood, milk, ink, grass, gravy, chocolate, egg, cheese, clay, pigment, and oil. One particularly preferred stain is a blood/milk/ink (BMI) stain.
The method can also involve incubating the stain with a cross-linking agent (e.g., hydrogen peroxide, bleaching agents, glutaraldehyde, and carbodiimides). The enzyme can be applied to the swatch in combination with a detergent ingredient. The method can additionally involve agitating the swatch and enzyme during incubation.
In still yet another embodiment, this invention provides methods of assaying for a preferred detergent composition. These methods involve providing a swatch of material comprising a piece of material and a stain; fixing the stain to the material; applying a detergent composition to the swatch; and incubating the watch and the detergent composition. The methods can additionally involve determining the degree of removal of the stain from the material. Preferred enzymes, materials and stains are as described herein. The method can involve incubating the stain with a cross-linking agent (e.g., hydrogen peroxide, bleaching agents, glutaraldehyde, and carbodiimides). The enzyme can be applied to the swatch in combination with the enzyme. In certain embodiments, the method involves agitating the swatch and detergent composition during incubation.
This invention also provides methods of determining the catalytic efficiency of an enzyme. The methods involve providing a swatch of material comprising a piece of material and a stain; applying the enzyme to the swatch; incubating the swatch and the enzyme; removing the swatch or supernatant; and measuring a constituent of the stain. Preferred enzymes, materials and stains are as described herein. The method can involve incubating the stain with a cross-linking agent (e.g., hydrogen peroxide, bleaching agents, glutaraldehyde, and carbodiimides). In certain preferred embodiments, the constituent is in d the supernatant. The constituent can be measured by determining its fluorescence and/or absorbance (e.g. absorbance spectra).
Also included herein are kits for the practice of the methods of this invention. One kit comprises a container containing a modified enzyme where one or more amino acid residues in the enzyme are replaced by cysteine residues, and the cysteine residues are modified by replacing the thiol hydrogen in the cysteine residues with a substituent group providing a thiol side chain comprising a multiply charged moiety. Another kit comprises a container containing a methane sulfonate reagent comprising a multiply charged substituent, and instructional materials teaching the use of the sulfonate reagent to couple a mutiply-charged moiety to a cysteine residue in a protein.
Definitons
The terms xe2x80x9cpolypeptidexe2x80x9d, xe2x80x9cpeptidexe2x80x9d and xe2x80x9cproteinxe2x80x9d are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term may also include variants on the traditional peptide linkage joining the amino acids making up the polypeptide.
The term xe2x80x9cresiduexe2x80x9d as used herein refers to natural, synthetic, or modified amino acids.
The terms enzyme includes proteins that are capable of catalyzing chemical changes in other substances without being permanently changed themselves. The enzymes can be wild-type enzymes or variant enzymes. Enzymes within the scope of the present invention include, but are not limited to, pullulanases, proteases, cellulases, amylases, isomerases, lipases, oxidases, oxidoreductases, hydrolases, aldolases, ketolases, glycosidases, oxidoreductases, hydrolases, aldolases, ketolases, glycosidases, lyases, ligases, transferases, and ligases.
The phrase xe2x80x9cmultiply-chargedxe2x80x9d or xe2x80x9cmultiple-chargexe2x80x9d refers to a net charge greater than +1 or less than xe2x88x921 at pH 7.0. A multiply charged substituent is a substituent that when covalently coupled to a subject enzyme bears a net charge greater than +1 or less than xe2x88x921 at pH 7.0.
A xe2x80x9cmutant enzymexe2x80x9d is an enzyme that has been changed by replacing an amino acid residue with a cysteine (or other) residue.
A xe2x80x9cchemically modifiedxe2x80x9d enzyme is an enzyme that has been derivatized to bear a substituent not normally found at that location in the enzyme.
A xe2x80x9cchemically modified mutant enzymexe2x80x9d or xe2x80x9cCMMxe2x80x9d is an enzyme in which an amino acid residue has been replaced with another amino acid residue (preferably a cysteine) and the replacement residue is chemically derivatized to bear a substituent not normally found on that residue.
The term xe2x80x9cthiol side chain groupxe2x80x9d, xe2x80x9cthiol containing groupxe2x80x9d, and thiol side chainxe2x80x9d are terms that can be used interchangeably and include groups that are used to replace the thiol hydrogen of a cysteine. Commonly the thiol side chain group includes a sulfur atom through which the thiol side chain group is attached to the thiol sulfur of the cysteine. The xe2x80x9csubstitutentxe2x80x9d typically refers to the group remains attached to the cysteine through a disulfide linkage formed by reacting the cysteine with a methanesulfonate reagent as described herein. While the term substituent preferably refers just to the group that remains attached (excluding its thiol group), the substituent can also refer to the entire thiol side chain group. The difference will be clear from the context.
The xe2x80x9cbinding site of an enzymexe2x80x9d consists of a series of subsites across the surface of the enzyme. The substrate residues that correspond to the subsites are labeled P and the subsites are labeled S. By convention, the subsites are labeled S1, S2, S3, S4, S1xe2x80x2, and S2xe2x80x2. A discussion of subsites can be found in Siezen et al. (1991) Protein Engineering, 4: 719-737, and Fersht (1985) Enzyme Structure and Mechanism, 2nd ed. Freeman, New York, 29-30. The preferred subsites include S1, S1xe2x80x2, and S2.
The terms xe2x80x9cstereoselectivityxe2x80x9d or xe2x80x9cstereoselectivexe2x80x9d when used in reference to an enzyme or to a reaction catalyzed by an enzyme refers to a bias in the amount or concentration of reaction products in favor of enantiomers of one chirality. Thus a stereoselective reaction or enzyme will produce reaction products that predominate in the xe2x80x9cDxe2x80x9d form over the xe2x80x9cLxe2x80x9d form (or xe2x80x9cRxe2x80x9d form over the xe2x80x9cSxe2x80x9d form) or conversely that predominate in the xe2x80x9cLxe2x80x9d form over the xe2x80x9cDxe2x80x9d form (or xe2x80x9cSxe2x80x9d form over the xe2x80x9cRxe2x80x9d form). The predominance of one chirality is preferably a detectable predominance, more preferably a substantial predominance, and most preferably a statistically significant predominance (e.g. at a confidence level of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably at least 98%).
The phrase xe2x80x9camino acid ##xe2x80x9d or xe2x80x9camino acid ## in the XX subsitexe2x80x9d is intended to include the amino acid at the referenced position (e.g. amino 156 of B. lentus subtilisin which is in the S1 subsite) and the amino acids at the corresponding (homologous) position in related enzymes.
A residue (amino acid) of a enzyme is equivalent to a residue of a referenced enzyme (e.g. B. amyloliquefaciens subtilisin) if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analagous to a specific residue or portion of that residue in B. amyloliquefaciens subtilisin (i.e., having the same or similar functional capacity to combine, react, or interact chemically).
In order to establish homology to primary structure, the amino acid sequence of the subject enzyme (e.g. a serine hydrolase) is directly compared to a reference enzyme (e.g. B. amyloliquefaciens subtilisin) primary sequence and particularly to a set of residues known to be invariant in all enzymes of that family (e.g subtilisins) for which sequence is known. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of the reference enzyme (e.g. B. amyloliquefaciens subtilisin) are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, (e.g., Asp32/His64/Ser221) should be maintained.
The conserved residues may be used to define the corresponding equivalent amino acid residues in other related enzymes. For example, the two (reference and xe2x80x9ctargetxe2x80x9d) sequences are aligned in to produce the maximum homology of conserved residues. There may be a number of insertions and deletions in the xe2x80x9ctargetxe2x80x9d sequence as compared to the reference sequence. Thus, for example, a number of deletions are seen in the thermitase sequence as compared to B. amyloliquefaciens subtilisin (see, e.g. U.S. Pat. No. 5,972,682). Thus, the equivalent amino acid or Tyr217 in B. amyloliquefaciens subtilisin in thermitase is the particular lysine shown beneath Tyr217.
The particular xe2x80x9cequivalentxe2x80x9d resides may be substituted by a different amino acid to produce a mutant carbonyl hydrolase since they are equivalent in primary structure.
Equivalent residues homologous at the level of tertiary structure for a particular enzyme whose tertiary structure has been determined by x-ray crystallography, are defined as those for which the atomic coordinates of 2 or more of the main chain atoms of a particular amino acid residue of the reference sequence (e.g. B. amyloliquefaciens subtilisin) and the sequence in question (target sequence) (N on N, CA on CA, C on C, and O on O) are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the enzyme in question to the reference sequence. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.   R  =                              ∑          h                ⁢                  "LeftBracketingBar"                      fo            ⁡                          (              h              )                                "RightBracketingBar"                    -              "LeftBracketingBar"                  fc          ⁡                      (            h            )                          "RightBracketingBar"                            ∑        h            ⁢              "LeftBracketingBar"                  fo          ⁡                      (            h            )                          "RightBracketingBar"            
Equivalent residues which are functionally analogous to a specific residue of a reference sequence (e.g. B. amyloliquefaciens subtilisin) are defined as those amino acids sequence in question (e.g. related subtilisin) which may adopt a conformation such that they will alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the reference sequence as described herein. Further, they are those residues of the sequence in question (for which a tertiary structure has been obtained by x-ray crystallography), which occupy an analogous position to the extent that although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of the reference sequence. The three dimensional structures would be aligned as outlined above. For an illustration of this procedure see U.S. Pat. No. 5,972,682.
A xe2x80x9cserine hydrolasexe2x80x9d is a hydrolytic enzyme utilizing an active serine side chain to serve as a nucleophile in a hydrolytic reaction. This term includes native and synthetic serine hydrolases as well as enzymes engineered to perform the reverse reaction, e.g., for synthetic purposes.
The xe2x80x9calpha/beta serine hydrolasesxe2x80x9d are a family of serine hydrolyases based on structural homology to enzymes including wheat germ serine carboxypeptidase""s II (see, e.g., Liam et al. (1992) Biochemistry 31: 9796-9812; Olli""s et al. (1992) Protein Engineering, 5: 197-211).
The xe2x80x9csubtilisin type serine proteasesxe2x80x9d refer to a family of serine hydrolyases based on structural homology to enzymes in including subtilisin BPNxe2x80x2 (Bott et al. (1988) J. Biol. Chem. 263: 7895-7906; Siezen and Louise (1997) Protein Science 6: 501-523). Subtilisin are bacterial or fungal proteases which generally act to cleave peptide bonds of proteins or peptides. As used herein, xe2x80x9csubtilisinxe2x80x9d means a naturally-occurring subtilisin or a recombinant subtilisin. A series of naturally-occurring subtilisins is known to be produced and often secreted by various microbial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus, is aspartate-histidine-serine. In the chymotrypsin related proteases, the relative order, however, is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases.
The xe2x80x9cchymotrypsin serine protease familyxe2x80x9d refers to a family of serine hydrolyases based on structural homology to enzymes including gamma chymotrypsin (Birktoft and Blow (1972) J. Molecular Biology 68: 187-240).
A xe2x80x9cdendritic polymerxe2x80x9d is a polymer exhibiting regular dendritic branching, formed by the sequential or generational addition of branched layers to or from a core. The term dendritic polymer encompasses xe2x80x9cdendrimersxe2x80x9d, which are characterized by a core, at least one interior branched layer, and a surface branched layer (see, e.g., Petar et al. Pages 641-645 In Chem. in Britain, (August 1994). A xe2x80x9cdendronxe2x80x9d is a species of dendrimer having branches emanating from a focal point which is or can be joined to a core, either directly or through a linking moiety to form a dendrimer. Many dendrimers comprise two or more dendrons joined to a common core. However, the term dendrimer is used broadly to encompass a single dendron.
Dendritic polymers include, but are not limited to, symmetrical and unsymmetrical branching dendrimers, cascade molecules, arborols, and the like, though the most preferred dendritic polymers are dense star polymers. The PAMAM dense star dendrimers (disclosed in U.S. Pat. No. 5,714,166) are symmetric, in that the branch arms are of equal length. The branching occurs at the hydrogen atoms of a terminal xe2x80x94NH2 group on a preceding generation branch. The lysine-based dendrimers are unsymmetric, in that the branch arms are of a different length. One branch occurs at the epsilon nitrogen of the lysine molecule, while another branch occurs at the alpha nitrogen, adjacent to the reactive carboxy group which attaches the branch to a previous generation branch.
Even though not formed by regular sequential addition of branched layers, hyperbranched polymers, e.g., hyperbranched polyols, may be equivalent to a dendritic polymer where the branching pattern exhibits a degree of regularity approaching that of a dendrimer.
A xe2x80x9cswatchxe2x80x9d is a piece of material (e.g. a natural or synthetic textile) that has a stain applied thereto. The material can be, for example, a fabric made of a natural fiber (e.g. cotton, hemp, wool), or a synthetic material (e.g. nylon, polyester, rayon, etc.) or a mixture of synthetic or natural fibers. The material need not be a textile, but can be any material subject to cleaning operations (e.g. glass, ceramic, Formica, etc.).
A xe2x80x9csmaller swatchxe2x80x9d is one that has been cut from the swatch of material either before or after fixing a stain to the swatch and can, for example, fit into the well of a 48 or 96 well microtiter plate. The xe2x80x9csmaller swatchxe2x80x9d can also be made by applying a stain to a smaller piece of material. Preferably the smaller swatch is about ⅝ inch in diameter, more preferably the smaller swatch is about 0.25 inches in diameter.
The phrase xe2x80x9creplacing the thiol hydrogen, in said one or more cysteine residuesxe2x80x9d does not require that every thiol hydrogen in every cysteine residue be replaced.